PCR-denaturing gradient gel electrophoresis and two feces antigen tests for detection of Helicobacter pylori in mice

PCR-denaturing Gradient Gel Electrophoresis (PCR-DGGE), a method suitable for the detection of microbial species in complex ecosystems, was evaluated for the detection and identification of Helicobacter spp. in feces and stomach tissue of mice. Two commercially available stool antigen tests for clinical diagnostics in humans were also evaluated in the C57Bl/6 mouse model of H. pylori infection. PCR-DGGE detected only Helicobacter ganmani in feces from H. pylori-infected as well as control animals, whereas in stomach specimens it demonstrated the presence of H. pylori in challenged and H. ganmani in control animals. Hence, the method detected DNA only of the predominant Helicobacter spp., which was also shown in cell dilution experiments. The Amplified IDEIA Hp StAR feces antigen test detected H. pylori in feces from all infected animals and generated no false-positive results, whereas the Premier Platinum HpSA-test also detected H. pylori in all infected animals but generated false-positive or equivocal results in 50% of the control animals. Premier Platinum HpSA, as opposed to Hp StAR, cross-reacted with non-pylori Helicobacter spp. in vitro.     

Magnetite nanoparticles can be coupled to microbubbles to support multimodal imaging

Microbubbles (MBs) are commonly used as injectable ultrasound contrast agent (UCA) in modern ultrasonography. Polymer-shelled UCAs present additional potentialities with respect to marketed lipid-shelled UCAs. They are more robust; that is, they have longer shelf and circulation life, and surface modifications are quite easily accomplished to obtain enhanced targeting and local drug delivery. The next generation of UCAs will be required to support not only ultrasound-based imaging methods but also other complementary diagnostic approaches such as magnetic resonance imaging or computer tomography. This work addresses the features of MBs that could function as contrast agents for both ultrasound and magnetic resonance imaging. The results indicate that the introduction of iron oxide nanoparticles (SPIONs) in the poly(vinyl alcohol) shell or on the external surface of the MBs does not greatly decrease the echogenicity of the host MBs compared with the unmodified one. The presence of SPIONs provides enough magnetic susceptibility to the MBs to accomplish good detectability both in vitro and in vivo. The distribution of SPIONs on the shell and their aggregation state seem to be key factors for the optimization of the transverse relaxation rate.     

LYP inhibits T-cell activation when dissociated from CSK

Lymphoid tyrosine phosphatase (LYP) and C-terminal Src kinase (CSK) are negative regulators of signaling mediated through the T-cell antigen receptor (TCR) and are thought to act in a cooperative manner when forming a complex. Here we studied the spatiotemporal dynamics of the LYP-CSK complex in T cells. We demonstrate that dissociation of this complex is necessary for recruitment of LYP to the plasma membrane, where it downmodulates TCR signaling. Development of a potent and selective chemical probe of LYP confirmed that LYP inhibits T-cell activation when removed from CSK. Our findings may explain the reduced TCR-mediated signaling associated with a single-nucleotide polymorphism that confers increased risk for certain autoimmune diseases, including type 1 diabetes and rheumatoid arthritis, and results in expression of a mutant LYP that is unable to bind CSK. Our compound also represents a starting point for the development of a LYP-based treatment of autoimmunity.     

Does Helicobacter pylori infection per se cause gastric cancer or duodenal ulcer? Inadequate evidence in Mongolian gerbils and inbred mice

A role for Helicobacter pylori infection in the development of gastric cancer in humans is well established; however, evidence for its carcinogenicity in animals remains inadequate. Mongolian gerbils and mice are commonly used to investigate the carcinogenicity of H. pylori, yet it is unclear whether H. pylori infection per se causes gastric cancer or duodenal ulcers in these animal models. Gastric adenocarcinoma in the gerbils was reported over 10 years ago, but this species has proved an unreliable model for studying H. pylori infection-associated gastric cancer. Helicobacter pylori infection alone appears insufficient to induce gastric cancer in these animals; additional carcinogenic insult is required. The development of invasive adenocarcinoma in inbred mice is rare regardless of the mouse or bacterial strain, and many long-term studies have failed to induce gastric cancer in these animals. Helicobacter pylori infection is also an established causative factor for duodenal ulcer in humans. However, few studies have attempted to develop animal models of H. pylori infection-induced duodenal ulcer. We therefore conclude that both Mongolian gerbils and inbred mice may be inadequate models for studying H. pylori infection-associated gastric cancer and that there is no animal model of H. pylori infection-induced duodenal ulcer.     

Population dynamics and production of the small copepod Oithona spp. in a subarctic fjord of West Greenland

The small cyclopoid copepod Oithona is widely occurring in polar areas; however, knowledge of its biology and ecology is very limited. Here, we investigate the population dynamics, vertical distribution, and reproductive characteristics of Oithona spp. from late winter to summer, in a subarctic fjord of West Greenland. During winter–early spring, the abundance of Oithona spp. was low (1.8 × 10³ ind. m^?2) and the population was mainly composed of late copepodites and adults, whereas in summer, abundance peaked and younger stages dominated (1.1 × 10⁶ ind. m^?2). In general, all stages of Oithona spp. remained in the upper 100 m, with nauplii exhibiting a shallower distribution. Although no general seasonal migration was found, a deeper distribution of the adult females in winter was observed. The mean clutch size of Oithona spp. varied from 16 to 30 eggs per female, peaking in summer. Egg production rates (EPR) were low in winter–early spring (0.13 ± 0.03 eggs female^?1 day^?1) and reached maximum values in summer (1.6 ± 0.45 eggs female^?1 day^?1). EPR of Oithona spp. showed a significantly positive relationship with both temperature and protozooplankton biomass, and the development of the population seemed to be appreciably affected by temperature. Oithona spp. remained active throughout the study, stressing the key importance of these small copepods in high-latitude ecosystems, especially in periods when larger copepods are not present in the surface layer.     

Improvement of the salt aggregation test to study bacterial cell-surface hydrophobicity

Abstract An improved salt aggregation test (improved SAT) was developed to sensitize the determination of bacterial cell-surface hydrophobicity. One drop of a fresh bacterial suspension standardized to an A ^1cm₅₄₀ of 20 (equivalent to 5 × 10⁹ cfu/ml), and one drop each of ammonium sulphate solutions stained with methylene blue, were mixed on a white hydrophobic paper card using toothpicks. The bacterial suspensions, methylene blue stock solutions and the ammonium sulphate solutions (0.01–4.0 M) were made in 0.02 M sodium phosphate buffer, pH 6.8. Bacterial aggregations were read immediately after mixing the salt/bacterial suspensions while the card was gently rocked. Readings were also confirmed the next day on dried preparations. The results proved independent of reading time and mixture conditions (wet or dry preparations). The improved SAT technique is very rapid and sensitive, the reaction is easily read with the naked eye, and the paper cards can be stored for documentation of aggregation patterns after drying. In the improved SAT, the Staphylococcus cells of different species aggregated in 5 ways: tiny, medial, flaky granular, particulated and macrofilamentous forms; Salmonella strains aggregated in flaky granular, particulated and macrofilamentous forms.     

Synthesis of glycoproteins in the Golgi complex of the mouse gallbladder epithelium during fasting,refeeding, and gallstone formation

Summary The mouse gallbladder epithelium was studied with light microscopic autoradiography and quantitative electron microscopy during fasting, refeeding and experimental gallstone formation. To determine the intracellular pathway of glycoproteins, H³-galactose was injected at different time intervals into the mice. At 10, 25 and 40 min after an intraperitoneal injection the gallbladders were fixed and prepared for light microscopy. As early as 10 min after injection, label was observed in supranuclear cytoplasmic regions and at 25 min, an increased radioactivity was present throughout the apical cytoplasm. At 40 min, silver grains were mainly present at the cell surface. Autoradiographs processed 25 min after an intraperitoneal H³-galactose injection after fasting for 48 h showed decreased supranuclear and apical radioactivity. After refeeding (12 h) there was an enhanced activity in both these regions. Animals fed a lithogenic diet for one month showed a marked increase of radioactive label mainly in cells of crypts and invaginations of the mouse gallbladder mucosa.Morphometric measurements of the Golgi apparatus revealed that deprivation of food significantly diminished the volume density of the Golgi apparatus. Refeeding the animals restored the volume density values to normal levels. In the course of gallstone formation there was a further significant increase in the volume density of the Golgi complexes as compared to controls.     

Cryptic domains of a 60 kDa heat shock protein of Helicobacter pylori bound to bovine lactoferrin

Abstract Bovine lactoferrin binds to a 60 kDa heat shock protein of Helicobacter pylori . Binding ability was related to human immunoglobulin G because bovine lactoferrin binding proteins were isolated by extraction of cell surface associated proteins with distilled water, applied on IgG-Sepharose and nickel sulphate chelate affinity chromatography. Binding was demonstrated by Western blot after purified protein was digested with α-chymotrypsin and incubated with peroxidase-labeled bovine lactoferrin. Binding was inhibited by bovine lactoferrin, lactose, rhamnose, galactose, and two iron-containing proteins, ferritin and haptoglobin. Helicobacter pylori binds ferritin and haptoglobin via charge or hydrophobic interactions because this binding was not inhibited by specific and various glycoproteins or carbohydrates. Carbohydrate moieties of bovine lactoferrin molecules seem to be involved in binding because glycoproteins with similar carbohydrate structures strongly inhibited binding. Scatchard plot analysis of the binding of peroxidase-labeled bovine lactoferrin to H. pylori cells yielded a k _d 2.88 × 10⁻⁶ M. In addition, binding of H. pylori cells to bovine lactoferrin was enhanced when bacteria treated with pepsin or α-chymotrypsin after isolation from iron-restricted and iron-containing media.